Publications by authors named "Bahareh Mohammadi"

24 Publications

  • Page 1 of 1

Rapid and sensitive UHPLC-DAD method for simultaneous determination of sofosbuvir and ledipasvir in human serum.

J Pharm Biomed Anal 2021 Feb 22;195:113860. Epub 2020 Dec 22.

Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran; Pharmaceutical Sciences Research Center, Kermanshah University of Medical, Sciences, Kermanshah, Iran. Electronic address:

Today, the direct-acting antiviral agents (DAAs) such as sofosbuvir (SOF) and ledipasvir (LED) are widely used to treat the hepatitis virus infection. The aim of this study was to develop a rapid, simple and valid method for simultaneous determination of SOF and LED in human plasma for bioavailability and pharmacokinetic studies. Chromatographic analysis was performed on the C column (Blue Orchid, 1.8 μm, 50 × 2 mm) using 0.1 % formic acid in water (pH 2.6) and acetonitrile (60:40; v/v) as mobile phase at a flow rate of 0.5 mL/min. The UV detector was set at 328 nm and 260 nm for analysis of SOF and LED, respectively. To 400 μL of plasma, 100 μL of clonazepam as the internal standard (I.S, 7 μg/mL) was added and the mixture subjected to liquid-liquid extraction using 1000 μL diethyl ether. The calibration curves were linear with coefficients of variation less than 8% for all analyses. The limit of quantification (LOQ) was 20 and 5 ng/mL for SOF and LED, respectively. The results of inter-day and intra-day precision showed good reproducibility and the total analysis time was 1.2 min. This method successfully applied for determination SOF and LED in four healthy volunteers.
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http://dx.doi.org/10.1016/j.jpba.2020.113860DOI Listing
February 2021

Protective and Therapeutic Effects of Aloe Vera Gel on Ulcerative Colitis Induced by Acetic Acid in Rats.

Clin Nutr Res 2020 Jul 30;9(3):223-234. Epub 2020 Jul 30.

Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah 671551616, Iran.

Ulcerative colitis (UC) is a chronic intestinal inflammation. Common clinical symptoms are weight loss, diarrhea, ulcers, and inflammation. Aloe vera (AV) has several medicinal properties including antioxidant, anti-inflammatory analgesic, and improvement of gastric and skin ulcers. This study aimed to investigate the protective and therapeutic effects of AV gel on acetic acid-induced UC in rats. UC was induced in 48 rats by injection of 4% acetic acid into the rectum. Protective and treatment groups received treatments 7 days before and after the induction of colitis, respectively. The negative control group, the positive control group, and AV groups received distilled water, sulfasalazine, and 50 and 300 mg/kg of gel extract, respectively. Water and food intake and body weight changes were recorded. The extent of the mucosal ulcers, colon tissue thickening, and mucosal bleeding were scored by the Gerald classification system score (microscopy observations). Slides of tissues were prepared for pathologic assay using the modified Wallace method (macroscopic observations). The results of the macroscopic and microscopic examination showed protective and therapeutic effects of 50 mg/kg dose of AV on acetic acid-induced colitis in rats which reduces the inflammation, ulcers and tissue damage compared with negative control (p < 0.05). There were no significant changes in the amount of water and food intake, body weight changes, and colon weight in protective and treatment groups. Based on the results, AV gel could be used to improve the symptoms of UC, as well as prevent people who are susceptible to the UC.
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http://dx.doi.org/10.7762/cnr.2020.9.3.223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402976PMC
July 2020

Protective effect of the isolated oligosaccharide from Rosa canina in STZ-treated cells through modulation of the autophagy pathway.

J Food Biochem 2020 Aug 6:e13404. Epub 2020 Aug 6.

Department of Life Sciences, National University of Kaohsiung, Kaohsiung, Taiwan.

Isolation of active components of therapeutic plants and discovering molecular mechanisms play a pivotal role in therapy of diabetes. This study aimed to determine the antidiabetic mechanism of an oligosaccharide isolated from Rosa canina (RCO) by measuring the expression of some miRNAs and their targets involved in autophagy. RCO was extracted and characterized by using HPLC and spectroscopic methods. Rin-5F cells were treated with STZ and RCO alone and in combination. The viability of the cells and the expression of miR-21, miR-22, Akt, ATG5, Beclin1, LC3A, and LC3B were analyzed using MTT assay, and qRT-PCR, respectively. Oligosaccharide fraction could improve the viability of RCO-treated cells as compared to STZ-treated cells. Further, the expression of autophagy markers was increased in RCO-treated diabetic cells compared to STZ-treated cells. The results indicated that the antidiabetic effects of the oligosaccharide components of R. canina seem to be mediated by modulation of autophagy pathway. PRACTICAL APPLICATIONS: Given effectiveness of an oligosaccharide fraction isolated from Rosa canina in management of diabetes in STZ-induced diabetic rats, we have intention to scrutinize its molecular mechanism as modulation of autophagy pathway in STZ-treated Rin-5F cells. It is expected that the results paved the way to speculate novel antidiabetic strategies.
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http://dx.doi.org/10.1111/jfbc.13404DOI Listing
August 2020

Anti-diabetic effect of a novel oligosaccharide isolated from Rosa canina via modulation of DNA methylation in Streptozotocin-diabetic rats.

Daru 2020 Dec 3;28(2):581-590. Epub 2020 Aug 3.

Molecular Pathology Research Center, Clinical Research Development Center, Imam Reza Hospital, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Background: Diabetes mellitus (DM) is a well-known clinical entity with various late complications. There is a surge of research aiming to use the medical herb in the management of DM.

Objective: This study aimed to investigate whether the alleviation of DM by an isolated compound from Rosa canina is mediated by DNA methylation in STZ-diabetic rats.

Methods: Sixty adult Wistar male rats were classified into control, diabetic and treatment groups. Rats were treated with STZ (40 mg/kg), metformin (500 mg/kg), and oligosaccharide fraction (OF; 10, 20 and 30 mg/kg) isolated from Rosa canina. DNA was extracted from the blood and pancreas to determine DNA methylation using the Global DNA Methylation kit. The expressions of DNA methyltransferases (Dnmts), PDX1, Ins1, GCK and PTP1B2 were determined by using qRT-PCR.

Results: The significant blood glucose-lowering potential of OF was associated with a reduced level of global DNA methylation (p < 0.05). The expression levels of Dnmts 1 and 3α increased in the pancreas and blood from diabetic rats compared to control group which declined by OF treatment (p < 0.05). Paradoxically, the expression of Dnmt 3β augmented in the pancreas and blood of OF group compared to diabetic ones (p < 0.05). Besides, the expressions of Pdx1, PTP1B2, Ins1 and GCK increased in OF-treated rats compared to diabetic groups.

Conclusion: Results revealed that DNA methylation plays a causal role in the effectiveness of the isolated OF. Furthermore, the possible regenerative potential of oligosaccharide in diabetic rats may have contributed to the modulation of DNA methylation.
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http://dx.doi.org/10.1007/s40199-020-00363-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704860PMC
December 2020

Characterization and anti-diabetic effects of the oligosaccharide fraction isolated from Rosa canina in STZ-Induced diabetic rats.

Carbohydr Res 2020 Mar 24;489:107927. Epub 2020 Jan 24.

Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Diabetes mellitus is the most common metabolic disorder characterized by chronic hyperglycemia. There has been a surge of research studies aiming to use natural products in the management of diabetes. The objective of this study was to isolate and characterize the structure and anti-diabetic mechanisms of the main ingredient from Rosa canina. The oligosaccharide was isolated from Rosa canina fruits and characterized by a combination of FTIR, NMR and Mass spectrometry. Wistar rats were divided into negative control, diabetic (type 2), isolated oligosaccharide (IO)-treated diabetic and positive diabetic controls. Oral glucose tolerance, gluconeogenesis and α-glucosidase inhibitory tests as well as immunohistochemistry and quantitative real time-PCR were performed to elucidate the molecular anti-diabetic mechanisms of IO. Structural analyses confirmed the oligosaccharide structure of isolated fraction. Gluconeogenesis and α-glucosidase activity were inhibited by IO in diabetic rats. The oral glucose tolerance test was improved significantly in the group treated with the IO (P < 0.05). Pancreatic β-cells and tissue pathological examination showed a significant improvement after the treatment period. In addition, the expression of Ngn3, Nkx6.1 and insulin increased in oligosaccharide-treated compared to untreated diabetic rats. Owing to the verified anti-diabetic effects and regenerative potential, isolated oligosaccharide could be considered as the promising drug in the management of diabetes.
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http://dx.doi.org/10.1016/j.carres.2020.107927DOI Listing
March 2020

Momordica charantia reverses type II diabetes in rat.

J Food Biochem 2019 11 23;43(11):e13021. Epub 2019 Aug 23.

Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Diabetes, a disease with abnormal production or use of insulin, is a growing concern that affects many individuals globally. Although many attempts have been made, there is no satisfactory treatment for diabetes. Recently, scientists have been exploring a promising treatment of diabetes involving herbal medicine. In this line, we show that Momordica charantia, a tendril-bearing vine belonging to the Cucurbitaceae family, permanently normalizes blood glucose levels comparable to healthy rats. Most importantly, M. charantia increases the expression of Insulin and Pdx1 genes while lowers the expression Glut2. Moreover, the number and size of the pancreatic islets have remarkably increased in treated animals. Liver ALT, AST, and ALP enzyme activities fell into normal range in treated animals suggesting the protective effect of M. charantia. These data indicate that M. Charantia improves the pancreas function by activating pancreatic beta cells and protecting liver tissue. PRACTICAL APPLICATIONS: Owing to the effectiveness of Momordica Charantia extracts in management of diabetes in STZ-induced diabetic rats, we have intention to evaluate the powder of Charantia to discover novel drug for treating diabetes. It is expected that the results could be translated in clinical trials.
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http://dx.doi.org/10.1111/jfbc.13021DOI Listing
November 2019

Effect of Stevia rebaudiana Bertoni extract on sexual dysfunction in Streptozotocin-induced diabetic male rats.

Cell Mol Biol (Noisy-le-grand) 2018 Feb 10;64(2):6-10. Epub 2018 Feb 10.

Department of Agronomy and Plant Breeding, Faculty of Agriculture, Razi University, Kermanshah, Iran.

Stevia rebaudiana Bertoni has been used locally as a non-calorie sweetener in medicine and diabetic diet which claimed to have aphrodisiac properties, although no scientific data of this function have been reported. The aim of this study was to investigate the effect of S. rebaudiana extract on sexual dysfunction, testosterone levels and number of Leydig cells in Streptozotocin (STZ)-induced diabetic male rats. A total of 28 diabetic male rats were randomly divided into 4 groups: diabetic group without any extract and 3 extract groups (5, 50 and 100 mg/kg). Seven normal control rats were treated with vehicle mount latency and frequency of (ML, MF), intromission latency and frequency (IL, IF), ejaculation latency and frequency of (EL, EF), the mount latency post ejaculation (MPE), the intromission latency post ejaculation (ILE), the intromission frequency post ejaculation (IFE) were recorded during 30 min on days 0, 14, 28. The serum testosterone levels, blood glucose, sex organs weight, number of leydig cells and histology of testicular tissue were measured. The stevia group (5 mg/kg) had a significant (p<0.05) increase in EF and IF. The number of Leydig cells in the diabetic group were significantly (p<0.05) reduced compared to the normal group and diabetic groups with extract (5 and 50 mg/kg). The serum testosterone levels and other sexual behaviors did not show any significant differences. The low- dose stevia extract with attention to antioxidant, vasodilator and anti-diabetic properties can be aphrodisiac in STZ- induced diabetic male rats.
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http://dx.doi.org/10.14715/cmb/2018.64.2.2DOI Listing
February 2018

Development and validation of a new HPLC-DAD method for quantification of sofosbuvir in human serum and its comparison with LC-MS/MS technique: Application to a bioequivalence study.

J Chromatogr B Analyt Technol Biomed Life Sci 2017 Sep 31;1063:118-122. Epub 2017 Aug 31.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran; School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran. Electronic address:

Although for many analyses tandem mass spectrometry (LC-MS/MS) systems have significant advantage over the high-performance liquid chromatography with diode array detection (HPLC-DAD) however, the HPLC methods are easier, cheaper and more available to perform. As no published method is available for quantitative HPLC analysis of sofosbuvir (SOF), an orally administered anti-hepatitis drug in human serum, this study was aimed to evaluate applicability of the HPLC technique to quantify sofosbuvir and comparison of the two methods for analytical performance. Following extraction of the drug and an internal standard (Hexobarbital), same chromatographic conditions were used for both the systems. After the chromatographic separation on a reverse phase C18 column using a mobile phase consisting of water (containing formic acid 0.5mL/L) and acetonitrile (57:43; v/v) at a flow rate of 0.8mL/min, the eluate was introduced into a DAD detector set at 261nm, then passed through the mass spectrometry system in single ion monitoring mode (SIM). For UV and mass spectrometry detections the calibration curves were linear over a concentration range of 25-3200 and 10-3200ng/mL, respectively and the linearity was over 0.998 for both the systems. Lower limit of quantification (LLOQ) for mass spectrometry and DAD detections were 10 and 25ng/mL, respectively. In conclusion sensitivity of DAD detection is sufficient enough to determine concentrations down to 0.5% of C which achieved in bioequivalence study of sofosbuvir and meet FDA requirements for these types of studies.
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http://dx.doi.org/10.1016/j.jchromb.2017.06.047DOI Listing
September 2017

Quantification of sofosbuvir in human serum by liquid chromatography with negative ionization mass spectrometry using the parent peak and its source-induced fragment: Application to a bioequivalence study.

J Sep Sci 2016 Jul 6;39(14):2702-9. Epub 2016 Jul 6.

School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran.

In the mass spectrometry of sofosbuvir, a new orally administered antihepatitis C drug, a weak peak is detected at the m/z value of the parent ion (m/z 530) as a result of in-source dissociation and current methods to its quantification, is based on monitoring of the parent peak using ultra high-performance liquid chromatography with tandem mass spectrometry. With these methods serum concentration of the drug is quantifiable only up to 4-5 h postdose. However, the fragmentation of the molecule generates a more stable ion at m/z 287 (base peak) with a signal intensity of about tenfold compared to the parent ion. Our study was aimed to improve sensitivity of analysis by acquisition of the m/z value of the daughter ion from which it originated instead of the parent molecule. This novelty allows us to measure serum concentrations of the drug for a longer time postdose and provides more opportunity for pharmacokinetic studies of sofosbuvir. Our method was linear over the concentration range of 2-2560 ng/mL of sofosbuvir in human serum with a limit of quantification of 2 ng/mL compared to 10 ng/mL reported previously. The coefficient variation values of both inter and intraday analysis were less than 13.8%, and the percentage error was less than 6.3.
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http://dx.doi.org/10.1002/jssc.201501375DOI Listing
July 2016

Antihyperlipidemic Effect of Syrian Mesquite (Prosopis farcta) Root in High Cholesterol Diet-Fed Rabbits.

J Evid Based Complementary Altern Med 2016 Oct 21;21(4):NP62-6. Epub 2016 Jan 21.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran Pharmaceutical Sciences Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Prosopis farcta root has been proposed as an efficacious natural drug for cardiovascular disorders in traditional medicine. The present study evaluates the efficacy of aqueous extract of Prosopis farcta root on experimental atherosclerosis development in rabbits with high cholesterol diet-induced hypercholesterolemia. Serum lipid parameters were significantly increased in the high cholesterol diet groups in comparison with the normal control group (P < .050). Histopathological findings revealed that atheromatous plaques were formed in both thoracic and abdominal aorta of hypercholestrolemic rabbits. Treatment with Prosopis farcta root significantly reduced total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein, and very low density lipoprotein levels compared to high cholesterol diet rabbits (P < .050). This finding may reflect a reduction of chest pain or the beneficial effects of this plant root extract on cardiovascular health. The present study can serve as a basis for future investigations on the other effects of this plant on cardiovascular health.
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http://dx.doi.org/10.1177/2156587215627552DOI Listing
October 2016

Applicability of LC-MS/MS to optimize derivatization of topiramate with FMOC-Cl using reacted/intact drug ratio.

J Chromatogr B Analyt Technol Biomed Life Sci 2013 Jun 26;928:32-6. Epub 2013 Mar 26.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Topiramate is an antiepileptic agent, which is structurally different from the other anticonvulsants. The drug has no UV-Vis absorption or emits fluorescence. Thus for its analysis using high performance liquid chromatography (HPLC) with conventional UV or fluorescence detectors, the drug should be derivatized with a suitable reagent. In previous study using fluorenylmethyl chloroformate (FMOC-Cl) and HPLC coupled with fluorescence detector, we reported an analytical method for derivatization and analysis of the drug in human serum. In this method, several factors including time and temperature of the reaction, pH and concentration of the used buffer, ratio of organic phase in the medium and removal of the reagent excess by glycine should be optimized to obtain maximum yield of the product. In HPLC coupled with fluorescence detector, there is not any signal from intact topiramate and only the final product (FMOC-topiramate) is appeared. Thus to optimize the reaction conditions for obtaining the highest derived yield, intensity of the final product peak is considered as a criteria for progression of the reaction. In LC-MS/MS system however, both free and reacted topiramate are visible in observed spectra. In the present study reaction of the drug with FMOC-Cl was re-optimized using LC-MS/MS technique on the basis of reacted/free topiramate ratio as the new and more accurate index. The results showed that, ratio of organic/aqueous phase has a dominant effect on the reaction, the most efficient temperature is 70°C and the reaction is reversed following addition of the glycine.
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http://dx.doi.org/10.1016/j.jchromb.2013.02.041DOI Listing
June 2013

9-fluorenylmethyl chloroformate as a fluorescence-labeling reagent for derivatization of carboxylic acid moiety of sodium valproate using liquid chromatography/tandem mass spectrometry for binding characterization: a human pharmacokinetic study.

J Chromatogr B Analyt Technol Biomed Life Sci 2012 Jan 30;880(1):12-8. Epub 2011 Nov 30.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.

In High Performance Liquid Chromatographic (HPLC) determination of chemicals with acidic functions, different labeling agents are used to improve sensitivity of the assay. 9-Fluorenylmethyl chloroformate (FMOC-Cl), on the other hand, is a suitable labeling agent, which reacts with both primary and secondary amines and less readily with hydroxyl groups in alkaline conditions. However, the reagent has not been applied in labeling of chemicals with acidic function yet. In this study which is the first report on application of FMOC-Cl in derivatization and analysis of a drug with acidic function, valproic acid (VPA), one of a series of fatty carboxylic acids with anticonvulsant activity, was derivatized using the reagent and quantified in serum samples by HPLC with fluorescence detection. In addition, to document the reaction between the labeling agent and carboxylic acid moiety of the drug, we developed a liquid chromatography-tandem MS/MS (LC-MS/MS) method. Following liquid-liquid extraction, derivatization of the drug and an internal standard was achieved in alkaline medium. The elute was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The present method is more sensitive comparing with other published HPLC procedures for analysis of VPA. The assay is sensitive enough to measure drug levels obtained in human single dose studies with a limit of quantification of 0.01 μg/mL. Also the method is linear over the concentrations range of 0.01-32 μg/mL of VPA in human serum using 100 μL serum sample and 5 μL injection. The coefficient variation values of both inter and intra day analysis were less than 12% and the percentage error was less than 4%. The method performance was studied and the validated procedure applied in a randomized cross-over bioequivalence study of two different VPA preparations in 24 healthy volunteers.
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http://dx.doi.org/10.1016/j.jchromb.2011.11.009DOI Listing
January 2012

Application of one-step liquid chromatography-electrospray tandem MS/MS and collision-induced dissociation to quantification of ezetimibe and identification of its glucuronated metabolite in human serum: A pharmacokinetic study.

J Chromatogr B Analyt Technol Biomed Life Sci 2010 Oct 21;878(28):2789-95. Epub 2010 Aug 21.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.

A new one-step liquid chromatography-electrospray tandem MS/MS method is described to quantify ezetimibe (EZM) a novel lipid lowering drug in human serum. Also using collision-induced dissociation (CID) of the analyte, identification and chromatographic separation of its major metabolite, ezetimibe glucuronide (EZM-G) is achieved in this study. A thawed serum aliquot of 100μL was deproteinated by addition of 500μL methanol containing omeprazole as internal standard (I.S.). Separation of the drug, its metabolite and the I.S. were achieved using acetonitrile-water (70:30, v/v) as mobile phase at flow rate of 0.5mL/min on a MZ PerfectSil target C18 column. Multiple reaction monitoring (MRM) mode of precursor-product ion transition (408.7→272.0 for EZM and 345→194.5 for the I.S.) was applied for detection and quantification of the drug while, EZM-G was chromatographically separated and identified using CID. The analytical method was linear over the concentration range of 1-32ng/mL of EZM in human serum with a limit of quantification of 1ng/mL. The coefficient variation values of both inter- and intra-day analysis were less than 8% whereas the percentage error was less than 3.7. The validated method was applied in a randomized cross-over bioequivalence study of two different EZM preparations in 24 healthy volunteers.
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http://dx.doi.org/10.1016/j.jchromb.2010.08.023DOI Listing
October 2010

Determination of oseltamivir carboxylic acid in human serum by solid phase extraction and high performance liquid chromatography with UV detection.

J Chromatogr B Analyt Technol Biomed Life Sci 2008 Mar 8;864(1-2):38-42. Epub 2008 Feb 8.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran; School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran.

This study was aimed at developing a fast and sensitive method for determination of oseltamivir carboxylic acid (OCA), the active moiety of anti-influenza agent, oseltamivir phosphate, in human serum by high performance liquid chromatography and UV detection. The analyte and an internal standard (vanillin) were extracted from human serum by a solid phase extraction (SPE) procedure. Chromatographic separation was achieved using a reverse phase C18 column with a mobile phase consisting of 0.05M phosphate buffer containing triethylamine (1mL/L; pH 3.0) and acetonitrile (70:30, v/v). The detection wavelength was set at 215nm. The average recoveries of the drug and internal standard were 98 and 85%, respectively. The calibration curve was linear over a concentration range of 15-6400ng/mL of OCA in human serum. The lower limits of detection and quantification were 5 and 15ng/mL, respectively. The coefficient variation values of both inter- and intra-day analysis were less than 12% whereas the percentage error was less than 4.5. The stability of the drug at the serum samples maintained at -40 degrees C for 60 days was found to be 100% from the initial value and no interferences were found from either endogenous components in serum or commonly co-administrated antiviral drugs. The validated method was applied to a randomized cross-over bioequivalence study of two different oseltamivir phosphate preparations in 24 healthy volunteers.
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http://dx.doi.org/10.1016/j.jchromb.2008.01.048DOI Listing
March 2008

High-performance liquid chromatographic determination of inactive carboxylic acid metabolite of clopidogrel in human serum: Application to a bioequivalence study.

J Chromatogr B Analyt Technol Biomed Life Sci 2008 Mar 8;864(1-2):168-72. Epub 2008 Feb 8.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.

A sensitive and rapid method is described for determination of clopidogrel carboxylic acid (CCA), the inactive metabolite of anti platelet agent, clopidogrel, in human serum. The analytical procedure involves liquid-liquid extraction of the analyte and an internal standard (phenytoin) with ethyl acetate. A mobile phase consisting of 0.05 M phosphate buffer containing triethylamine (0.5 mL/L; pH 5.7) and acetonitrile (56:44 v/v) was used and chromatographic separation was achieved using C18 analytical column at detector wavelength of 220 nm. The calibration curves were linear over a concentration range of 0.05-10 microg/mL of CCA in human serum. The total run time of analysis was 5.5 min and the lower limits of detection (LOD) and quantification (LOQ) were 0.02 and 0.05 microg/mL, respectively. The method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. The validated method was applied in a randomized cross-over bioequivalence study of two different clopidogrel preparations in 24 healthy volunteers.
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http://dx.doi.org/10.1016/j.jchromb.2008.01.049DOI Listing
March 2008

Rapid and sensitive bioanalytical method for measurement of fluvoxamine in human serum using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent: application to a human pharmacokinetic study.

J Chromatogr B Analyt Technol Biomed Life Sci 2007 Oct 3;857(2):322-6. Epub 2007 Aug 3.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.

A sensitive and rapid high-performance liquid chromatographic method for the analysis of fluvoxamine, a selective serotonin reuptake inhibitor in human serum, is described using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent. The drug and an internal standard (fluoxetine) were extracted from 0.25 mL of serum using ethyl acetate as extracting solvent and subjected to pre-column derivatization by the reagent. A mobile phase consisting of methanol and sodium phosphate buffer (0.05 M; pH 2.8) containing 1 mL/L triethylamine (72:28 v/v) was used and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm x 4.6mm) column. The fluorescence derivatives of the drugs were monitored at excitation and emission wavelengths of 470 and 537 nm, respectively. The calibration curve was linear over the concentration range of 0.5-240 ng/mL with a limit of quantification (LOQ) of 0.5 ng/mL using 0.25 mL serum sample. The method validation was performed for its selectivity, specificity, sensitivity, precision and accuracy. In this method, which was applied in a randomized cross-over bioequivalence study of two different fluvoxamine preparations in 24 healthy volunteers, the sensitivity and run time of analysis were significantly improved.
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http://dx.doi.org/10.1016/j.jchromb.2007.07.044DOI Listing
October 2007

Determination of clarithromycin in human serum by high-performance liquid chromatography after pre-column derivatization with 9-fluorenylmethyl chloroformate: application to a bioequivalence study.

J Chromatogr B Analyt Technol Biomed Life Sci 2007 May 30;850(1-2):417-22. Epub 2006 Dec 30.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.

A sensitive liquid chromatographic method for the analysis of clarithromycin, a macrolide antibiotic, in human serum using pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) is described. The method involved liquid-liquid extraction of the drug and an internal standard (amantadine) followed by pre-column derivatization of the analytes with FMOC-Cl. A mixture of 0.05 M phosphate buffer containing triethylamine (2 mL L(-1); pH 3.8) and methanol (17:83, v/v) was used as mobile phase and chromatographic separation was achieved on a Shimpack CLC-ODS column. The eluate was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The analytical method was linear over the concentration range of 0.025-10 microg mL(-1) of clarithromycin in human serum with a limit of quantification of 0.025 microg mL(-1). The assay is sensitive enough to measure drug levels obtained in human single dose studies. In the present method, sensitivity and run time of analysis have been improved, and successfully applied in a bioequivalence study of three different clarithromycin preparations in 12 healthy volunteers.
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http://dx.doi.org/10.1016/j.jchromb.2006.12.027DOI Listing
May 2007

A novel high sensitivity HPLC assay for topiramate, using 4-chloro-7-nitrobenzofurazan as pre-column fluorescence derivatizing agent.

J Chromatogr B Analyt Technol Biomed Life Sci 2007 May 17;850(1-2):400-4. Epub 2006 Dec 17.

Medical Biology Research Center, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran.

A new, sensitive and simple high-performance liquid chromatographic method for analysis of topiramate, an antiepileptic agent, using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent is described. Following liquid-liquid extraction of topiramate and an internal standard (amlodipine) from human serum, derivatization of the drugs was performed by the labeling agent in the presence of dichloromethane, methanol, acetonitrile and borate buffer (0.05 M; pH 10.6). A mixture of sodium phosphate buffer (0.05 M; pH 2.4): methanol (35:65 v/v) was eluted as mobile phase and chromatographic separation was achieved using a Shimpack CLC-C18 (150 x 4.6 mm) column. In this method the limit of quantification of 0.01 microg/mL was obtained and the procedure was validated over the concentration range of 0.01 to 12.8 microg/mL. No interferences were found from commonly co-administrated antiepileptic drugs including phenytoin, phenobarbital carbamazepine, lamotrigine, zonisamide, primidone, gabapentin, vigabatrin, and ethosuximide. The analysis performance was carried-out in terms of specificity, sensitivity, linearity, precision, accuracy and stability and the method was shown to be accurate, with intra-day and inter-day accuracy from -3.4 to 10% and precise, with intra-day and inter-day precision from 1.1 to 18%.
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http://dx.doi.org/10.1016/j.jchromb.2006.12.016DOI Listing
May 2007

An isocratic high performance liquid chromatographic method for quantification of mycophenolic acid and its glucuronide metabolite in human serum using liquid-liquid extraction: application to human pharmacokinetic studies.

Clin Chim Acta 2006 Aug 6;370(1-2):185-90. Epub 2006 Mar 6.

Medical Biology Research Center, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Background: Mycophenolate mofetil (MMF) is a morpholinoethyl ester of mycophenolic acid (MPA), which is widely used as an immunosuppressive agent in renal transplant patients for the prophylaxis of acute rejection. We describe a fast and sensitive isocratic HPLC method for simultaneous quantification of the immunosuppressant mycophenolic acid, and its major metabolites in human serum.

Methods: The analytes were extracted from serum using ethyl acetate/2 propanol (4-1, v/v) and subjected to an isocratic HPLC method using a phenyl analytical column. A mobile phase consisted of methanol-0.05 mol/l sodium phosphate buffer (46/54 v/v; pH 2.5) containing hexadecyl trimethylammonium bromide (100mg/l w/v) and triethylamine (0.25% v/v) was used.

Results: The standard curve was linear from 0.050 to 51.2 microg/ml and 0.125 to 64 microg/ml, for mycophenolic acid and its metabolite, respectively. The method showed excellent selectivity, specificity, sensitivity, precision and accuracy. The respective limits of quantification for the drug and its metabolite were 0.050 and 0.125 microg/ml. This method was applied in a bioequivalence study following single dose administration of 2 different mycophenolate mofetil preparations in 24 healthy volunteers. Blood samples were analyzed and pharmacokinetic parameters of mycophenolic acid and its metabolite were compared.

Conclusion: This procedure is simple, and comparing to the previously published methods, more sensitivity is obtained and less time is needed for sample preparation. Less time was needed for the liquid-liquid extraction. Although, suitability of the method has been demonstrated in pharmacokinetic studies of MMF in normal subjects, its specificity in renal and hepatic transplant patients has not been established and the stated upper linearity of MPAG may not be sufficient for use in transplant patients.
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http://dx.doi.org/10.1016/j.cca.2006.02.017DOI Listing
August 2006

Sensitive microanalysis of gabapentin by high-performance liquid chromatography in human serum using pre-column derivatization with 4-chloro-7-nitrobenzofurazan: Application to a bioequivalence study.

J Chromatogr B Analyt Technol Biomed Life Sci 2006 Jun 27;837(1-2):24-8. Epub 2006 Apr 27.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Most of the published methods for analysis of gabapentin, an antiepileptic agent, in human serum require automated o-phthalaldehyde derivatization of the drug and immediate injection of the unstable derivatives formed. A new, very sensitive and simple high-performance liquid chromatographic method for quantitation of the drug in human serum using 4-chloro-7-nitrobenzofurazan (NBD-Cl) as a fluorescent labeling agent is presented. In this method the sensitivity was significantly improved and the limit of quantification of 0.002 microg/ml was obtained using 100 microl serum sample and 10 microl injection. However, the LOQ can be improved by increasing the sampling volume. The procedure involved protein precipitation of serum by acetonitrile followed by derivatization with NBD-Cl. Amlodipine was used as internal standard and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm x 4.6 mm) column. The fluorescence derivative of the drug was monitored at excitation and emission wavelengths of 470 and 537 nm, respectively. A mobile phase consisting of methanol and sodium phosphate buffer (0.05 M; pH 2.5) containing 1 ml/l triethylamine (65:35, v/v) was used. The calibration curve was linear over the concentration range of 0.002-15 microg/ml. No interferences were found from commonly co-administrated antiepileptic drugs. The method was applied in a randomized cross-over bioequivalence study of two different gabapentin preparations in 24 healthy volunteers.
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http://dx.doi.org/10.1016/j.jchromb.2006.03.056DOI Listing
June 2006

A new on-line, in-tube pre-column derivatization technique for high performance liquid chromatographic determination of azithromycin in human serum.

J Chromatogr B Analyt Technol Biomed Life Sci 2006 Jan 23;830(2):355-8. Epub 2005 Nov 23.

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran; School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Pre-column derivatization methods for high performance liquid chromatographic assay of specific pharmaceutical agents using 9-fluorenylmethyl chloroformate (FMOC-Cl) have received special attention because highly fluorescent and stable adducts are provided by these methods. However, unlike the post-column on-line techniques, long derivatization time is needed and the reaction cannot be well controlled. A new, sensitive and fast pre-column on-line derivatization technique coupled with high-performance liquid chromatography using FMOC-Cl as labeling agent is described and validated for determination of azithromycin in human serum. After extraction of the drug from serum, the residue was reconstituted in mixture of acetonitrile-phosphate buffer (3:1, v/v; pH 8.5) and directly injected onto the chromatographic system. Continuous on-line derivatization and analysis of the compounds were successfully performed using in-tube elution of FMOC-Cl. The total time needed for derivatization and chromatographic analysis of the drug was 13 min. The assay was reliable and reproducible, with limit of quantification of 10 ng/ml. The described technique may offer significant advantages over existing off-line derivatization methods using FMOC-Cl.
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http://dx.doi.org/10.1016/j.jchromb.2005.10.044DOI Listing
January 2006

Determination of atorvastatin in human serum by reversed-phase high-performance liquid chromatography with UV detection.

J Chromatogr B Analyt Technol Biomed Life Sci 2005 Nov 16;826(1-2):41-5. Epub 2005 Sep 16.

Medical Biology Research Center, Medical School, Karmanshah University of Medical Sciences, Kermanshah 6714869914, Iran; School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran.

A rapid and sensitive high-performance liquid chromatographic method was validated and described for determination of atorvastatin in human serum. Following liquid-liquid extraction of the drug and an internal standard (sodium diclofenac), chromatographic separation was accomplished using C18 analytical column with a mobile phase consisting of sodium phosphate buffer (0.05 M, pH 4.0) and methanol (33:67, v/v). Atorvastatin and the internal standard were detected by ultraviolet absorbance at 247 nm. The average recoveries of the drug and internal standard were 95 and 80%, respectively. The lower limits of detection and quantification were 1 and 4 ng/ml, respectively, and the calibration curves were linear over a concentration range of 4-256 ng/ml of atorvastatin in human serum. The analysis performance was studied and the method was applied in a randomized cross-over bioequivalence study of two different atorvastatin preparations in 12 healthy volunteers.
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http://dx.doi.org/10.1016/j.jchromb.2005.08.008DOI Listing
November 2005

High-performance liquid chromatographic determination of lamivudine in human serum using liquid-liquid extraction; application to pharmacokinetic studies.

J Chromatogr B Analyt Technol Biomed Life Sci 2005 Sep;823(2):213-7

Medical Biology Research Center, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran.

A simple, fast, and sensitive high performance liquid chromatographic (HPLC) assay was developed for quantitation of lamivudine in human serum. Lamivudine is polar compound and its extraction from the human serum in previously published HPLC methods involved either protein precipitation or solid phase extraction techniques. However, existence of endogenous peaks which interfere with the drug or appeared as late eluting peaks and lead to long run time of analysis has been reported. Application of either an ion pairing agent in the mobile phase or time consuming column purge has been used in the published methods. Present paper describes liquid - liquid extraction of lamivudine and internal standard (famotidine) using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent and salting out approach. The mobile phase was a mixture of phosphate buffer (0.05 M) containing triethylamine (1 mL/L, v/v; pH 3.5) and methanol (91:9, v/v) at a flow rate of 2.2 mL/min. The analysis was performed on a column (150 mm x 6 mm i.d.) which was packed with 5 microm particles of ODS packing material. Under these conditions no interference in the assay from any endogenous substance was observed. The limit of quantification was evaluated to be 5 ng/mL. Accuracy and precision of the method were also studied and the technique was shown to be selective and linear into the concentration range of 5-2500 ng/mL. This method has been used in two randomized crossover bioequivalence studies of 100 and 150 mg lamivudine preparations in 12 and 24 healthy volunteers, respectively.
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http://dx.doi.org/10.1016/j.jchromb.2005.06.044DOI Listing
September 2005

High performance liquid chromatographic determination of topiramate in human serum using UV detection.

J Chromatogr B Analyt Technol Biomed Life Sci 2005 Aug;822(1-2):322-5

Medical Biology Research Center, Medical School, Kermanshah University of Medical Sciences, Kermanshah 6714869914, Iran.

Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid-liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 microg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992.
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http://dx.doi.org/10.1016/j.jchromb.2005.05.032DOI Listing
August 2005